Development of a neutralization assay for Nipah virus using pseudotype particles.

Abstract

Nipah virus (NiV) and Hendra virus (HeV) are zoonotic paramyxoviruses capable of causing severe disease in humans and animals. These viruses require biosafety level 4 (BSL-4) containment. Like other paramyxoviruses, the plaque reduction neutralization test (PRNT) can be used to detect antibodies to the surface glycoproteins, fusion (F) and attachment (G), and PRNT titers give an indication of protective immunity. Unfortunately, for NiV and HeV, the PRNT must be performed in BSL-4 containment and takes several days to complete. Thus, we have developed a neutralization assay using VSV pseudotype particles expressing the F and G proteins of NiV (pVSV-NiV-F/G) as target antigens. This rapid assay, which can be performed at BSL-2, was evaluated using serum samples from outbreak investigations and more than 300 serum samples from an experimental NiV vaccination study in swine. The results of the neutralization assays with pVSV-NiV-F/G as antigen showed a good correlation with those of standard PRNT. Therefore, this new method has the potential to be a rapid and cost-effective diagnostic method, especially in locations that lack high containment facilities, and will provide a valuable tool for basic research and vaccine development.

Pseudotype particles are can serve as antigens in an enzyme immunoassay. Panel A shows reactivity of HMAF prepared against NiV with pVSV-NiV-F/G and pVSV. Plates were coated with dilutions of the pseudotype particles as indicated before being tested by enzyme immunoassay as described in Materials and Methods. Panel B shows the reactivity of pseudotype pVSV-NiV-F/G with positive (Hu+) and negative (Hu−) human serum samples and samples from mice vaccinated with recombinant vaccinia viruses expressing either NiV F and G (anti-rvv/NiV-FG) or measles H and F (anti-rvv/ChiH-EdF).

Antibody responses following experimental vaccination for NiV. Figure shows results of the RIP assay. Antigens were prepared by infecting Vero cells with vaccinia viruses expressing NiV F and NiV-G and labeling with 35S-methionine as described in Materials and Methods. Representative serum samples from pigs that had been vaccinated with recombinant canarypoxvirus vaccines expressing NiV glycoproteins, CP-F, CP-G, or CP-F and CP-G at 0 (D0), 7 (D7), 14 (D14), 21 (D21), 28 (D28), and 49 (D49) dpv were used. Lanes C1 and C2 show reactions with mock infected and labeled Vero cells and C3 shows reaction with HMAF prepared to NiV. RIP assays were performed as described in the Materials and Methods.

Analysis of serum samples from NiV infected humans by RIP and pseudotype neutralization assay. Panel A shows immunoprecipitation of radio labeled NiV F and G (expressed from recombinant vaccinia virus) with human serum samples collected during outbreaks of NiV. Samples H737, H713, H700, H699 and H677 were from NiV-infected individuals; samples H675, H661, H658, H680 and H622 were from uninfected contacts. The controls include a serum sample from a volunteer in the laboratory (Neg), a positive control human serum sample from the initial outbreak of NiV in Malaysia (Hum+), and HMAF to NiV. Panel B shows the ability of the serum samples to inhibit luciferase activity in Vero cells infected with pVSV-NiV-F/G. Serum samples were tested at four fold dilutions beginning at 1:40 and the infections and luciferase assays were performed as described in the Materials and Methods. Negative controls samples are indicated by dashed lines and NiV positive samples have solid lines.

Comparison of the results of PRNT with the neutralization assays using pVSV-NiV-F/G as antigen. Graphs show geometric mean titers (GMT) from both assays (pVSV-NiV-F/G in top graph; PRNT in lower graph) for serum collected 0, 7, 14, 21, 28, 35, 42 and 49 dpv (pigs receiving high dose vaccine only are shown).

Sensitivity and specificity of the pseudotype neutralization assays compared to PRNT. Figure shows comparison tables of the results from the neutralization assay using VSV-NiV-F/G and PRNT. Serum samples were collected at 0, 7, 14, 21, 28, 35, 42 and 49 dpv. All animals were boosted on day 21. Results from pigs receiving high dose and low dose vaccinations are included.